The long-term objective of this project is to establish the molecular functions of DEAD box proteins. These RNA dependent ATPases are critical to most cellular processes involving RNA including translational initiation, mRNA splicing, and assembly of RNA-protein complexes such as the ribosome. Th model system for these studies will be the E. coli protein DbpA, which is unique among DEAD box proteins in that it displays high affinity and specificity for 23S rRNA. The specific aims of this project are 1) a thorough biophysical characterization of the specific enzyme-substrate complex and 2) a detailed understanding of the molecular consequences of ATP hydrolysis in terms of conformational changes in DbpA and the RNA substrate. Size exclusion chromatography and analytical ultracentrifugation will provide information on stoichiometries and RNA-protein crosslinking will identify domains important to substrate specificity. Questions of functionality will be addressed through in vitro helicase assays, fluorescence energy transfer experiments, and differential scanning calorimetry. Quantitative analysis of the enzymology of DbpA will lead to new insights for the mode of action of this important class of proteins.